Colony stimulating factor-1 (CSF-1) is an important chemoattractant for osteoclasts, the cells that mediate bone loss in periodontal disease. CSF-1 is released by osteoblasts and stromal cells in response to IPS, TNF and other cytokines and proresorptive hormones. Cell motility involves prominent cytoskeletal changes, and we have found that CSF-1 induces rapid cytoplasmic spreading, actin reorganization and motility in osteoclasts. We have identified a CSF-1-induced signaling cascade in osteoclasts in which Rac and cofilin are key downstream messengers. This application will clarify the distal components of the signaling cascade that connect activated Rac and cofilin to the actin cytoskeleton. Osteoclasts express two Rac isoforms, Rac-1 and Rac-2. Recent work has highlighted the non-redundant roles of these two isoforms. We will selectively delete Rac-1 and Rac-2 in osteoclasts to explore their separate contributions to CSF-1's effects on mature osteoclast function and motility. One key downstream pathway from activated Rac is through LIM-kinase and cofilin. We have shown that microinjecting a neutralizing antibody to cofilin completely blocks CSF-1-induced actin remodeling. Activation of cofilin involves a rapid phosphorylation/ dephosphorylation cycle called phosphocycling. We hypothesize that either LIM kinase-1 or LIM kinase-2 phosphorylates cofilin and that Slingshot is a candidate phosphatase for cofilin. We will use cell-based assays and knock-out mice to interrogate this signaling cascade in CSF-1-treated osteoclasts. Rac has recently been shown to target activated alphaVbeta3 integrin to the leading edge of motile cells and we will examine this function in CSF-1-activated osteoclasts. Finally, we have identified delta-COP, a transport vesicle-coating protein, and murine BCA3 as unique Rac-interacting partners in osteoclasts. We have confirmed these interactions in vitro and in vivo in osteoclasts. We hypothesize that the Rac delta-COP interaction is critical for Golgi reorientation during migration and for vesicular trafficking in osteoclasts. We will explore the role and regulation of delta-COP and mBCAS in osteoclasts using cell and molecular biological approaches. These studies will better define the mechanism by which CSF-1 regulates mature osteoclasts. Understanding the cellular basis for bone loss is critical to improving dental health and identifying potential targets for drug discovery.